This application is based on PCT Application No. PCT/AU96/00258, filed May 2, 1996; which is a continuation of Australian Patent Application No. PN 2742, filed May 2, 1995.
The present invention relates to a previously unidentified erbB receptor target designated GDU. The present invention relates to a polynucleotide encoding GDU and to methods of detecting the presence of GDU.
Many intracellular targets for receptor tyrosine kinases (RTKs) contain one or more src homology (SH)2 domains. These are conserved, non-catalytic domains of approximately 100 amino acids which bind to short peptide sequences containing phosphotyrosine (Cohen et al, Cell 80, 237-248, 1995). Since receptor autophosphorylation on specific tyrosine residues follows RTK activation, SH2 domains mediate receptor-substrate, as well as other protein-protein interactions, during signal transduction. SH2 domains contain not only a pocket lined with basic residues which binds the phosphotyrosine but also an additional binding pocket or groove which interacts with amino acids C-terminal to this residue, this determining the specificity of the interaction. The particular autophosphorylation sites present on a given RTK therefore define the SH2 domain-containing signalling proteins that it can recruit and hence, to a large extent, the signalling specificity of the receptor. SH2 domains are often accompanied in signalling proteins by two other conserved protein modules; SH3 domains, which bind to proline-rich peptide ligands, and pleckstrin-homology (PH) domains. The function of the latter remains ill-defined, and both protein and phospholipid ligands have been described.
SH2 domain-containing proteins can be divided into two classes (Schlessinger and Ullrich Neuron, 9,383-301 1992); Class I, which also possess a catalytic function e.g. phospholipase C-xcex31 (PLC-xcex31) and the GTPase activating protein for Ras (Ras-GAP), and Class II, which contain only non-catalytic protein modules and are thought to function as adaptors, linking separate catalytic subunits to receptors or other signalling proteins e.g. Grb2. The tissue expression of particular SH2 domain-containing proteins varies from ubiquitous, e.g. Grb2, which performs a fundamental role in linking tyrosine kinases to Ras signalling, to relatively restricted e.g. Grb7, which is mainly expressed in the liver and kidney (Margolis et al Proc. Natl. Acad.
Sci. USA, 89, 8894-8898, 1992). Presumably the latter protein performs relatively specialised signalling functions. The CORT (cloning of receptor targets) technique, in which cDNA expressionlibranes are screened with the tyrosine phosphorylated C-terminus of the EGF receptor represents a powerful methodology for the identification and characterisation of novel, SH2 domain-containing, receptor substrates (Skolnik et al Cell 65, 83-90, 1991).
Members of the erbB family of RTKs and their ligands are implicated both in normal mammary gland development and the growth and progression of human breast cancer. Furthermore, marked alterations in the expression or activity of several SH2 domain-containing proteins have been observed in human breast cancers or breast cancer-derived cell lines, suggesting that this represents an additional level at which RTK signalling may be modulated in this disease (Daly, Breast Cancer Res Treat, 34, 85-92, 1995). We therefore chose normal human mammary epithelial cells as a basis for a CORT screening program and hence identification of novel, and relatively tissue specific, erbB receptor targets.
Screening of a HMEC 184 xcexEXlox cDNA library isolated 1 Ras-GAP, 2 Grb2 cDNAs and a cDNA encoding a novel SH2 domain-containing protein. This protein, designated GDU or Grb14 (the designations xe2x80x9cGDUxe2x80x9d and xe2x80x9cGrb14xe2x80x9d are used interchangeably herein), is related both in molecular architecture and sequence homology to Grb7 and Grb10, previously identified erbB receptor targets. These three proteins also share significant sequence homology, over an approximately 300 amino acid region encompassing the PH domain, with the C. elegans gene F10E9.6. The latter gene has recently been shown to encode a protein (mig. 10) critical for longitudinal neuronal migration in C. elegans; members of the Grb7 gene family, including GDU, may therefore be involved in the regulation of cell migration in higher organisms.
Analysis of GDU gene expression in normal breast epithelial cells and a large series of human breast cancer cell lines revealed that expression was limited predominantly to normal breast cells and the more highly differentiated, estrogen receptor positive, breast cancer cell lines. Also, GDU mRNA was overexpressed in the DU-145 prostate carcinoma cell line relative to the normal prostate and two other prostate cancer cell lines. GDU may therefore serve as a prognostic indicator and/or a tumour marker in both breast and prostate cancer. Furthermore, since altered expression of GDU may contribute to the abnormal proliferation, invasion and/or migration of cancer cells, GDU signal transduction may provide a novel therapeutic target in human cancer. Finally, since GDU is involved in downstream signalling initiated by the platelet derived growth factor receptor (PDGFR), it may provide a target in diseases or conditions in which PDGF plays a regulatory role e.g. wound healing, fibrotic conditions, atherosclerosis.
In a first aspect the present invention consists in a polynucleotide encoding GDU, the polynucleotide having a sequence which encodes a polypeptide having an amino acid sequence as shown in FIG. 2 or a sequence which hybridises thereto.
In a preferred embodiment of the present invention the polynucleotide has a sequence as shown in FIG. 2.
In a second aspect the present invention consists in a polypeptide, the polypeptide having an amino acid sequence as shown in FIG. 2.
In a third aspect the present invention consists in an antibody which binds to the polypeptide of the second aspect of the present invention.
The antibody may be monoclonal or polyclonal, however, it is presently preferred that the antibody is a monoclonal antibody.
In a fourth aspect, the present invention consists in an oligonucleotide probe of at least 12 nucleotides, the oligonucleotide probe having a sequence such that the probe selectively hybridises to the polynucleotide of the first aspect of the present invention under stringent conditions.
In a preferred embodiment of this aspect of the present invention the oligonucleotide is labelled. In a further preferred embodiment of the present invention the oligonucleotide is of at least 18 nucleotides.
In a fifth aspect the present invention consists in method of detecting the presence of GDU in a sample, the method comprising reacting the sample with an antibody of the second aspect of the present invention or a oligonucleotide probe of the fourth aspect of the present invention and detecting the binding of the antibody or the probe.